Difference between revisions of "Part:BBa K729004:Experience"

Revision as of 03:51, 19 September 2015

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Applications of BBa_K729004

Testing the compatibility of the nuclease with Azo-dyes

The BBa_K729004 periplasmic nuclease that UCL iGEM 2012 submitted and characterised was able to demonstrate positive DNase agar assays. This assay involves washing DNA-containing agar with HCl after streaking bacteria. After this wash, a ‘halo’ surrounding the streaked bacteria indicates that extracellularly secreted DNase digested the DNA surrounding the colonies within the agar. In order to further characterise this BioBrick and incorporate it into our project as a biosafety method of minimising the transfer of extracellular DNA, we decided to test whether BBa_K729004 would function in the presence of Azo-Dyes.

Figure 1 - BBa_K729004 BBa_K729004 periplasmic nuclease enzyme shows functionality in the presence of multiple Azo-dyes. Figure showing that the BBa_K729004 periplasmic nuclease enzyme is still able to digest the surrounding DNA in the DNase agar. Figure 1a and 1b demonstrate the presence of halos around colonies on plates with and without Acid Orange 7 (AO7) azo-dye. Figure 1c and 1d demonstrate the presence of halos around colonies on plates with and without Reactive Black 5 (RB5). All azo-dye agar plates were made with a 1:500 dilution of 0.5mgml-1 dye.

Since the azo-dye degradation would be taking place in industrial environments, being the products of the bioreaction at one point dumped into other channels of water-treatment or into the environment, it is essential that there are certain barriers that stop the DNA from our genetically modified organisms from potentially being transferred into wild-type strains. On a basic first-level defense, this assay suggests that horizontal gene transfer could be inhibited in an azo-dye contaminated environment byBBa_K729004 , as it has proven effective in degrading extracellular bacterial DNA.

By UCL 2014

Using BBa_K729004 to degrade biofilms

We ordered this part from the Registry to test if Staphylococcal DNase is capable of degrading E. coli biofilms. We received the part on 23rd July 2015 from iGEM HQ.

We used PCR to add on a BspHI site upstream of the coding sequence, and subsequently ligated it into the commercial expression vector pBAD/HisB so that we can use L-arabinose to induce the expression of this gene.

We cloned the BBa_K729004[pBAD] plasmid into E. coli MG1655 and using arabinose induction showed that:

- The staphylococcal DNase encoded by this part is successfully secreted into the extracellular media

- The staphylococcal DNase encoded by this part can degrade biofilms

By Oxford 2015

User Reviews

UNIQ72bcfc5acdd01430-partinfo-00000001-QINU UNIQ72bcfc5acdd01430-partinfo-00000002-QINU

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 '''By UCL 2014'''
+===Using BBa_K729004 to degrade biofilms===
+We ordered this part from the Registry to test if Staphylococcal DNase is capable of degrading ''E. coli'' biofilms. We received the part on 23rd July 2015 from iGEM HQ.
+We used PCR to add on a BspHI site upstream of the coding sequence, and subsequently ligated it into the commercial expression vector pBAD/HisB so that we can use L-arabinose to induce the expression of this gene.
+We cloned the BBa_K729004[pBAD] plasmid into ''E. coli'' MG1655 and using arabinose induction showed that:
+- The staphylococcal DNase encoded by this part is successfully secreted into the extracellular media
+- The staphylococcal DNase encoded by this part can degrade biofilms
+'''By Oxford 2015'''
 ===User Reviews===